Emerging evidence has shown that the capacity of a tumor to grow and propagate is dependent on a small subset of cells within a tumor. This concept was originally based on the observation that when cancer cells of many different types were assayed for their proliferative potential in various in vitro or in vivo assays, only a minority of cells were able to proliferate extensively. This observation gave rise to the idea that malignant tumors are comprised of a small subset of distinct cancer stem cells which have great proliferative potential, as well as more differentiated cancer cells, which have very limited proliferative potential. Our research group has identified a highly tumorigenic cancer cell population within human pancreatic cancers that functions as a cancer stem cell. The goal of this proposal is to examine protein expression in this subpopulation of pancreatic cancer stem cells as an approach that may help us develop more effective therapies. This cancer stem cell population represents a small number of cells so that specialized micro-proteomic approaches will be needed to profile changes in protein expression and identification related to cell signaling pathways. The methods used will involve a shotgun approach where the whole cell lysates will be digested and the peptides separated using capillary isolectric focusing in the pH dimension. The proteins will then be further fractionated using nonporous RP-HPLC and identified based upon LC-MS/MS of the peptides against protein databases where we expect to identify around 2500 proteins. The active signal pathways will then be studied using bioinformatic methods and the KEGG pathway database using the protein profiles obtained. The signal pathways identified by proteomics will then be compared to data from oligonucleotide arrays to obtain corresponding genomic data. We will use these methods specifically to search for dysregulated pathways related to the Sonic Hedgehog(SHH) pathway and also for other pathways such as Wnt, Notch and Bmi-1. PUBLIC HEALTH RELEVANCE The proposed research project may provide a methodology for studying the proteomics of small numbers of pancreatic cancer stem cells and may further facilitate identification of key signaling pathways that may be dysregulated relative to the non-stem cell population. These protein pathways may be responsible for the unique properties of stem cells such as their proliferative potential, as well as resistance to therapeutic interventions (including both drug and radiation treatment). Greater understanding of the signaling pathways involved in pancreatic cancer stem cell growth and regulation may provide critical information for development of therapeutic modalities specifically targeting the cancer stem cell population relative to the non-cancer stem cell population.